Clinical trials consisting of (1) CTLA-4 blockade with a monoclonal antibody and (2) adoptive T cell therapy with in vitro expanded autologous T cells have demonstrated dramatic anti-tumor responses in a susbset of patients. These therapies have laid the foundation for the development of more effective immunotherapy strategies to provide greater benefit to patients. In our studies we are focused on identifying the mechanisms that underlie the anti-tumor responses seen after anti-CTLA-4 and adoptive T cell therapy. Pertinent to this application, we were the first to investigate the role of the inducible costimulator (ICOS) molecule during anti-CTLA-4 therapy. We demonstrated that some patients had a significant increase in T cells expressing ICOS, and that a sustained increase in ICOS+ T cells correlated with clinical benefit. ICOS is a T cell specific molecule and a member of the CD28 family. ICOS is upregulated on T cells only after activation. It has only one known ligand referred to as ICOS-ligand (ICOSL). The ICOS/ICOSL pathway is thought to affect T cell responses via ICOS-mediated PI3-kinase signaling but a role for this in tumor immunity has not been established. Furthermore, prior to our studies, the ICOS/ICOSL pathway had not been reported to play a role in murine or human anti-tumor responses. We investigated the role of the ICOS/ICOSL pathway in anti-tumor responses using mouse models and demonstrated that the ICOS/ICOSL pathway plays an essential role in tumor immunity in the setting of CTLA-4 blockade. Anti-CTLA-4 therapy led to an increased frequency of CD4+ICOS+ and CD8+ICOS+ T cells in tumor- bearing wild-type mice. ICOS+ T cells predominantly produced Th1 cytokines including the known anti-tumor Th1 cytokine IFN- . We further demonstrated that the ICOS/ICOSL pathway was essential for optimal antigen- specific T cell proliferation and cytokine production. Most importantly, anti-tumor responses induced by CTLA-4 blockade were significantly impaired in ICOS-knockout (KO) and ICOSL-KO mice. This data establishes that ICOS is not a mere marker of T cell activation but is involved in the anti-tumor effect induced by anti-CTLA-4. We have thus provided the first direct evidence that the ICOS/ICOSL pathway is required for optimal anti-tumor responses in the setting of CTLA-4 blockade. We now hypothesize that the ICOS/ICOSL pathway induces optimal T cell anti-tumor responses due to ICOS-mediated PI3K signaling. We further hypothesize that the ICOS/ICOSL pathway can be targeted to improve anti-tumor responses in the setting of CTLA-4 blockade, as well as adoptive T cell therapy. Therefore, in the current proposal, we aim to: 1) Elucidate the role of ICOS-mediated PI3-kinase signaling in T cell anti-tumor responses; 2) Improve anti-tumor responses by targeting the ICOS/ICOSL pathway in combination with CTLA-4 blockade; and 3) Establish the relevance of the ICOS/ICOSL pathway in the setting of adoptive T cell therapy. PUBLIC HEALTH RELEVANCE: Inducible costimulator (ICOS) and its ligand (ICOSL) have not previously been explored as targets for immunotherapy strategies in cancer patients. Based on our published and preliminary data, the ICOS/COSL pathway plays an important role in anti-tumor immune responses in the setting of CTLA-4 blockade, which implicates this pathway as a novel target for cancer immunotherapy strategies that involve combination therapy with CTLA-4 blockade or other immune-based therapies such as adoptive T cell therapy. The studies proposed in this grant application will allow us to obtain pre-clinical data that will facilitate future clinical trials targeting the ICOS/ICOSL pathway for patient benefit.